Strongly quenching oligomeric excimer/quencher pairs for detection schemes
View Patent ↗Compositions and systems are provided for the high efficiency quenching small water-soluble oligomers, or oligofluors, of from about 1-10 kd in size, where the oligofluors comprise multiple excimeric or exciplex forming fluorophores arranged on a scaffold, which are efficiently quenched by a quencher entity linked to the oligomer through a cleavable moiety. Fluorophores of interest include, without limitation, aromatic fluorophores such as pyrenes, e.g. benzopyrene, perylene, pyrene, etc. In some embodiments the oligofluor/quencher combination provides for a Stern-Vollmer constant (K SV ) of greater than about 10 6 M −1 , and may be greater than about 10 7 M −1 , greater than about 10 8 M −1 , or more. In some embodiments of the invention, the scaffold is a phosphodiester/glycoside backbone, e.g. an analog of a polynucleotide. The system of oligofluors and quenchers can be used in qualitative and quantitative screening and detection methods to detect any enzymatic, chemical or catalytic activity that can cleave the moiety between the quencher and scaffold.
1. A sensor having the structure:
wherein A is a backbone group or a linker, and may be absent or present;
X is a backbone group;
R is a fluorochrome, where each R can be the same or different, and wherein at least two R groups are an excimer or exciplex forming fluorochrome;
n is 2 to 20,
Y is a cleavable linker, and
Z is a quencher for R, wherein Z quenches with a Stern-Vollmer constant (K sv ) of greater than 10 6 M −1 , wherein the increase in fluorescence, following cleavage of the quencher, is at least about 20-fold.
2. The sensor of claim 1 , wherein the increase in fluorescence is at least about 100-fold.
3. The sensor of claim 1 , wherein X is a phosphodiester, phosphorothioate, phosphotriester, locked nucleic acid (LNA); morpholino; 2′-O-methyl RNA, or peptide nucleic acid.
4. A sensor having the structure:
wherein A is a backbone group or a linker, and may be absent or present;
X is a backbone group;
R is a fluorochrome, where each R can be the same or different, and wherein at least two R groups are an excimer or exciplex forming fluorochrome;
n is 2 to 20,
Y is a cleavable linker, and
Z is a quencher for R, wherein R is selected from pyrene, perylene, benzopyrene, oxoperylene, rubrene, perylene bisimide, styrene, anthracene, tetracene, pentacene, and fluorene.
5. The sensor of claim 1 , wherein Z is selected from methyl viologen, methyl red, dabcyl, dabsyl, dansyl, FRET acceptors, TAMRA, Iowa black, nitroxyl quenchers, black hole quenchers, dimethylaminostilbene, dimethylaminoazobenzene, dimethylaniline, nitrobenzene, pentafluorobenzene, methylpyridinium, and phenyl-(methylpyridinium).
6. The sensor of claim 1 , wherein Y is directly or indirectly cleavable by an analyte selected from lipase, esterase, nuclease, peptidase, glycosidase, metal catalyst or environmental condition.
7. The sensor of claim 1 , having homogenous R groups.
8. The sensor of claim 1 , having mixed R groups.
9. The sensor of claim 1 , linked to a substrate through A.
10. The sensor of claim 1 , linked to a member of a specific binding pair through A.
11. A method for detection of an analyte of interest, the method comprising:
contacting a sample suspected of comprising said analyte of interest with the sensor according to claim 1 , wherein Y is directly or indirectly susceptible to cleavage by the analyte; and
measuring the fluorescence of the sample.
12. The sensor of claim 1 , wherein Y comprises a chain of less than about 30 atoms in length, wherein said chain comprises a cleavable bond; and one or more of an amide, a thioether and a triazole.
13. The sensor of claim 12 , wherein Y is of the structure:
—C 1 -T 1 -C 2 -B-T 2 -
wherein C 1 and C 2 are each independently a chain of about 2 to 6 atoms in length;
wherein T 1 is selected from a thioether group, a triazole group, and an amide group;
wherein T 2 is selected from a single bond, a chain of about 2 to 6 atoms in length, an aminomethylene group, an aminomethylphenyl group, and a benzyl group; and
wherein B is a cleavable bond.
14. The sensor of claim 1 , wherein X is a phosphodiester such that the backbone groups Xn comprise a deoxyribosephosphate backbone.
15. A sensor having the structure:
wherein A is a backbone group or a linker, and may be absent or present;
X is a backbone group;
R is a fluorochrome, where each R can be the same or different, and wherein at least two R groups are an excimer or exciplex forminq fluorochrome;
n is 2 to 20,
Y is a cleavable linker, and
Z is a quencher for R, wherein Z quenches with a Stern-Vollmer constant (K sv ) of greater than 10 6 M −1 .